The rats were orally administered 50, 100, and 250 mg/kg of quercetin suspended in 0.5% Na-carboxymethylcellulose orally for seven consecutive days. On the 8th day, the proximal 20 cm of the small intestine was extirpated following intraperitoneal injection of Zoletil® (25 mg/kg of zolazepam and 25 mg/kg of tiletamine). Total RNA was extracted from the scraped intestinal mucosa using RNAiso (Takara, Tokyo, Japan) according to the manufacturer’s protocol. Subsequently, mRNA was reverse transcribed to cDNA using the Takara RNA PCR Kit (AMV ver. 3.0) and a Takara PCR Thermal Cycler Dice TP650. Quantitative PCR was performed in a LightCycler 1.5 (Roche Diagnostics, Mannheim, Germany). Thermocycling for each reaction was performed in a final volume of 20 μL reaction mixture using SYBR Green Premix Ex Taq (Takara Bio Inc., Otsu, Japan) and the primers listed in Table 1 . The mRNA level was estimated by relative quantification to 18s RNA.
Takara pcr thermal cycler dice tp650
Manufactured by Takara Bio
Sourced in Japan
The Takara PCR Thermal Cycler Dice TP650 is a laboratory equipment designed for performing polymerase chain reaction (PCR) experiments. It is capable of precisely controlling temperature and cycling parameters required for DNA amplification.
Automatically generated - may contain errors
Lab products found in correlation
Rnaiso, by Takara Bio (2 mentions)
Lightcycler 1, by Roche (2 mentions)
Sybr green premix ex taq, by Takara Bio (1 mentions)
Takara rna pcr kit amv ver 3, by Takara Bio (1 mentions)
Rna pcr kit amv ver 3, by Takara Bio (1 mentions)
Primescript high fidelity rt pcr kit, by Takara Bio (1 mentions)
Fastgene rna basic kit, by Nippon Genetics (1 mentions)
3 protocols using takara pcr thermal cycler dice tp650
1
Quercetin Intestinal Absorption Protocol
2
Quantifying BCRP Expression in Rats
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BCRP/rat BCRP mRNA was quantified in the cells and tissues of rats according to a previous report [24 (link)]. Briefly, LS174T cells were seeded at a density of 2 × 105 cells/mL, followed by incubation in 5, 10, and 50 μM quercetin in 0.5% DMSO for 48 h with replacement every 24 h. Total RNA was extracted using RNAiso (Takara, Tokyo, Japan) and reverse transcribed to cDNA using the Takara RNA PCR™ Kit (AMV ver. 3.0) and Takara PCR Thermal Cycler Dice TP650. Quantitative PCR was performed using the LightCycler 1.5 (Roche Diagnostics, Mannheim, Germany). The mRNA levels of BCRP were calculated by relative quantification using the mRNA levels of glyceraldehyde-3-phosphate dehydrogenase.
Male SD rats were administered quercetin suspended in 0.5% sodium carboxymethyl cellulose at doses of 50, 100, and 250 mg/kg for seven consecutive days and liver, kidney, and small intestinal tissues were excised on the 8th day. RNA extraction and quantitative PCR were performed as described above. The mRNA level of Bcrp was calculated by relative quantification with 18s RNA.
Male SD rats were administered quercetin suspended in 0.5% sodium carboxymethyl cellulose at doses of 50, 100, and 250 mg/kg for seven consecutive days and liver, kidney, and small intestinal tissues were excised on the 8th day. RNA extraction and quantitative PCR were performed as described above. The mRNA level of Bcrp was calculated by relative quantification with 18s RNA.
3
Gene Expression Analysis by RT-PCR
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Cells were washed twice with PBS, and the total RNA was extracted using the FastGene RNA Basic kit (Nippon Genetics, Tokyo, Japan). The concentration and quality of total RNA were calculated from the measured absorbance. Reverse transcription (RT)-PCR was performed using the PrimeScript High Fidelity RT-PCR Kit and TaKaRa PCR Thermal Cycler Dice TP 650 (Takara Bio) for 500 ng of total RNA. Annealing was performed at 65 C for 5 minutes. Reverse transcription to cDNA was performed at 42 C for 30 minutes, followed by incubation at 95 C for 5 minutes to stop the enzyme reaction. The cDNA was amplified by PCR using the following primers: integrin a6: forward, 5 0 -GGCCTTATGAAGTTGGTGGA-3 0 and reverse, 5 0 -AGAGGCCAGACCTTCTCCAT-3 033 ; CD71: forward, 5 0 -GAGGAGCCAGGAGAGGACTT-3 0 and reverse, 5 0 -ACGCCAGACTTTGCTGAGTT-3 034 ; and GAPDH: forward, 5 0 -TGCACCACCAACTGCTTAGC-3 0 and reverse, 5 0 -GGCATGGACTGTGGTCATGAG-3 0 . 35 PCR was performed for 30 cycles at 98 C for 10 seconds, 55 C for 5 seconds, and 72 C for 1 minute.
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